Flaviviruses are positive-stranded RNA, arthropod-borne viruses. Nearly half of the 70 known flaviviruses are associated with human disease and many of these regularly cause significant human morbidity and mortality. St. Louis encephalitis, dengue and West Nile virus (WNV), have infected humans during recent outbreaks in the United States. Little is currently known about the molecular mechanisms utilized by flaviviruses to initiate and regulate RNA synthesis or translation. Predicted conserved viral RNA sequence and structure elements and interactions with particular cell proteins as well as viral nonstructural proteins may play essential roles in regulating these processes. Although the conserved 3' SL and adjacent CS1 sequence in the flavivirus genomic RNA have been shown to be essential for virus replication, little is known about the individual functional elements contained in this region. We propose to test the cis-activity of individual nts within predicted conserved 3' RNA structures and sequences by mutation in a WNV infectious clone and to also test the relevance of predicted alternative RNA tertiary structures. The cis-activity of individual nts in three fine mapped binding sites for the cell protein, EF-1alpha, previously shown to interact with this region of the viral RNA will also be tested. An in vitro translation system and an autonomous WNV uncoupled replicon will be used to assess the participation of individual cis-acting nts in the regulation of viral RNA translation and/or replication, respectively. The proposed studies will provide new insights about the regulatory mechanisms that control flavivirus RNA translation and replication as well as about the participation of cell protein interactions in these processes. The proposed studies will also identify novel targets for designing attenuated recombinant candidate live vaccines and for the development of novel antiviral strategies.